Solutions and films of glycated chitosan

ABSTRACT

Preparations containing glycated chitosan in a physiologically compatible carrier are described. Viscoelastic preparations comprise 1.0-12.0 percent by weight of glycated chitosan in an aqueous solution while lower viscosity solutions possess 0.1-0.5 percent by weight glycated chitosan. The otherwise free amino groups of the chitosan polymer are preferably 30-90% glycated and possess an average molecular weight of between 100,000 and 2,000,000 Daltons, and, most preferably between 100,000 and 1,000,000 Daltons.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to viscous medical preparations, and,more specifically, to viscous solutions containing glycated chitosan anddried films prepared therefrom.

2. Background

Viscoelastic substances are used routinely in the medical field forprotection and manipulation of tissues and maintenance of spaces.Viscoelastic materials prepared from various naturally occurringsubstances or synthesized in the laboratory include sodium hyaluronate,chondroitin sulfate and combinations thereof, cellulosic materials, andpolymers based on acrylamide. Previously used viscoelastics havedisadvantages which include allergic reactions, neurotoxic impurities,inadequate viscosity or viscoelasticity; unacceptable levels ofparticulate materials; gels or bulky polymer chains which might enterand plug fluid meshworks; procurement difficulties due to the use ofanimal derived raw materials; and excessive cost. Materials which arenot broken down and absorbed in vivo also generally require that they beirrigated from the body.

It is thus an object of the present invention to provide improvedviscoelastic preparations which are far less subject to the above-noteddisadvantages.

SUMMARY OF THE INVENTION

In connection with the present invention there are provided preparationscontaining glycated chitosan in a physiologically compatible carrier.Such preparations possess useful viscoelastic properties, are absorbableby hydrolysis in vivo and, surprisingly, are endowed with antibacterialqualities. Dry films prepared from glycated chitosan solutions are alsoprovided.

Chitosan is a deacetylated derivative of chitin, a plentiful substancereadily isolated from the shells of crustaceans such as crab, lobsterand shrimp. Glycated chitosan refers to products resulting from thereaction between free amino groups of chitosan and carbonyl groups ofreducing monosaccharides and/or oligosaccharides.

The preferred viscoelastic preparations comprise 1.0-12.0 percent byweight of glycated chitosan in an aqueous solution, depending upon thedesired physical properties. The otherwise free amino groups of thechitosan polymer are preferably 30-90% glycated, and the moleculespossess an average molecular weight of between 100,000 and 2,000,000Daltons, and, most preferably between 100,000 and 1,000,000 Daltons. Theinventive viscoelastic solutions are of a viscosity greater than 10,000centistokes (one centistoke=a resistance to flow of 1 mm/sec) and, mostpreferably between 10,000 and 80,000 centistokes, measured at 25° C. Aparticularly preferred form of glycated chitosan for use in theinventive preparations is galacto-chitosan.

The inventive solutions and films are useful in a myriad of medicalapplications, including, for example, as viscoelastic ophthalmic agentsand as drug carriers for medicaments for topical application to the eye;antimicrobial coatings for use on adhesive strips or burn and wounddressings; tissue void replacement materials; lavage solutions; tissueseparation films, mouth rinses; facial lotions; or as hand and bodydisinfectants.

In an alternate embodiment, certain low viscosity preparations areprovided for use as ophthalmic wetting agents for topical application tothe eye and as drug carriers for medicaments for topical application tothe eye. The preparations are particularly useful when combined withcertain medicaments or drugs, which are entrapped in the glycatedchitosan preparations. Since the preparations are broken down over timeby enzymes in eye secretions, they have the characteristic of sustainedrelease. For use as a wetting agent or eye drop, the inventivepreparations comprise less than 1.0, and preferably 0.1-0.5, percent byweight of glycated chitosan in a physiologically compatible aqueoussolution, while possessing 30-90%, preferably 60%, glycation of theotherwise free amino groups of the chitosan polymer and having aviscosity in the range from about 10 centistokes to about 100centistokes.

Especially for ophthalmic preparations, the glycated chitosan ispreferably contained in an aqueous solution having a pH between 5.5 and7.5, and, most preferably, in a dilute slightly acidic aqueous solutionhaving a pH between 6.3 and 7.

A better understanding of the present invention, its several aspects,and its objects and advantages will become apparent to those skilled inthe art from the following detailed description, wherein there isdescribed the preferred embodiment of the invention, simply by way ofillustration of the best mode contemplated for carrying out theinvention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Before explaining the present invention in detail, it is important tounderstand that the invention is not limited in its application to thedetails of the particular preparations and the steps described herein.The invention is capable of other embodiments and of being practiced orcarried out in a variety of ways. It is to be understood that thephraseology and terminology employed herein is for the purpose ofdescription and not limitation.

Chitin is a linear homopolymer composed of N-acetylglucosamine unitsjoined by β 1→4 glycosidic bonds. Chitin and chitosan (resulting fromvarious degrees of deacetylation of chitin), and their derivatives, areendowed with interesting chemical and biological properties that haveled to a varied and expanding number of industrial and medicalapplications, including their use in connection with bandages andsutures, burn dressings, skin substitutes, bone and dental prostheses,food packaging, drug encapsulation, cosmetics, metal chelation andassociated antioxidant effects, waste water treatment, hemostasis,anticoagulants (after sulfation), and dye doping, among other things.

Solubilization of chitin and chitosan has been achieved by partialhydrolysis to shorten the length of their molecules. For chitosan,treatment with a variety of acids, both organic and inorganic, leads tothe formation of water soluble chitosonium salts by protonation of thefree amino groups. Additional modifications of the amino groups includethe introduction of chemical groups such as carboxymethyl, glyceryl,N-hydroxybutyl and others. U.S. Pat. No. 5,093,319, incorporated hereinby reference, describes the use of chitin and chitosan derived polymerssuch as chitosan acetate; chitosan lactate; chitosan sulfate; chitosanglutamate; methyl-chitosan; N-carboxyl methyl-chitosan; O-carboxylmethyl-chitosan; N,O-carboxyl ethyl-chitosan; N-carboxyl ethyl-chitosan;O, carboxyl ethyl-chitosan; N,O-carboxyl propyl-chitosan; N-carboxylpropyl chitosan; O-carboxyl ethyl chitosan; cross-linked chitosan orderivatives thereof, and carboxyl alkyl chitins such as carboxymethylchitin, carboxyethyl chitin and carboxypropyl chitin in a viscoelasticform for in vivo use as a biodegradable adhesion preventative.

Glycated chitosan was first described in U.S. Pat. No. 5,747,475,incorporated herein by reference, as an immunoadjuvant in connectionwith laser/sensitizer assisted immunotherapy for cancer. (See Cols. 8-18of the '475 patent). Glycated chitosan is a product of the glycation(i.e., non-enzymatic glycosylation) of free amino groups of chitosan,followed by stabilization by reduction. Glycation endows the chitosanwith advantageous solubility characteristics which facilitated the useof this chitosan derivative in conjunction with laser/sensitizerassisted immunotherapy. The glycation of chitosan also renders thechitosan more hydrophilic whereby more water is absorbed and retained bythe polymer than would otherwise be the case.

The products resulting from the reaction between free amino groups ofchitosan and carbonyl groups of reducing monosaccharides and/oroligosaccharides (i.e., glycated chitosan) are mainly a mixture ofSchiff bases, i.e. the carbon atom of the initial carbonyl group doublebonded to the nitrogen atom of the amino group, and Amadori products,i.e. the carbon atom of the initial carbonyl group bonded to thenitrogen atom of said amino group by a single bond while an adjacentcarbon atom is double bonded to an oxygen atom forming a ketone group.The products may be used as such or after stabilization by reductionwith hydrides, such as sodium borohydride, or by exposure to hydrogen inthe presence of suitable catalysts. The galactose derivative of chitosanis particularly preferred insofar as galactose has a relatively highnaturally occurring incidence of its open chain form. The glycatedchitosan may be initially prepared in a powder form, as a viscoussolution, or in other forms. But as used in connection with theinventive preparations herein, the glycated chitosan is in aqueoussolution or is dried into a thin film from such an aqueous solution.

Preparation of Glycated Chitosan

Glycated chitosan may be obtained by reacting chitosan with astoichiometric excess of a monosaccharide and/or oligosaccharide,preferably in the presence of an acidifying agent, for a time sufficientto accomplish Schiff base formation between the carbonyl group of thesugar and the primary amino groups of chitosan (also referred to hereinas glycation of the amino group) to a degree whereby a 30-90% (and mostpreferably 60%) glycation of the chitosan polymer is achieved. Asmentioned above, this may be, and is preferably, followed bystabilization by reduction of Schiff bases and of their rearrangedderivatives (Amadori products). The molecular weight of the polymerspreferred for use in the present invention most preferably range from100,000 to 1,000,000 Daltons. NMR tracings can be used to verify thebonding of the monosaccharides and/or oligosaccharides to the chitosanpolymer, whereas chemical measurement of remaining free amino groups,such as via a ninhydrine reaction, can be used to assess the degree ofglycation.

The inventive solutions contain glycated chitosan in a physiologicallycompatible carrier. “Physiologically compatible” is employed to refer tomaterials which, when in contact with tissues in the body, are notharmful thereto. The term is intended in this context to define, but isnot limited to, aqueous solutions which are approximately isotonic withthe physiological environment of interest. Non-isotonic solutionssometimes may be clinically useful such as, for example dehydratingagents. Additional components of the inventive solutions may includevarious salts such as NaCl, KCl, CaCl₂, MgCl and Na based buffers.Especially for ophthalmic and viscosurgical preparations, theconcentrations of the components of the inventive solutions may bederived to have an osmolality on the order of 250-350 and are bufferedto maintain the desired pH of from 5.5 to 7.5. The concentration of thesalts and the buffering agents may be chosen to be similar to that ofcommercially available intraocular irrigating solutions and viscoelasticsurgical fluids.

Acceptable manners of preparation of the inventive glycated chitosansolutions and films in accordance with the several aspects of thepresent invention will be further understood with reference to thefollowing non-limiting examples. The inventive preparations possessing aconcentration greater than 1 wt. % glycated chitosan are obtained byconcentrating, through dialysis or other means, a 1 wt. % stock. For thelow viscosity inventive solutions, a 1 wt. % stock solution is diluted.The viscosity of the inventive solutions may be manipulated by varyingeither the concentration of the glycated chitosan in solutions, theaverage molecular weight of the glycated chitosan molecules, or thestructure of the glycated chitosan molecule.

EXAMPLE 1

An initial 1 wt. % aqueous solution of glycated chitosan was prepared asfollows:

(a) 1 gram of chitosan was added to 100 ml of de-ionized water in aflask and mixed with magnetic stirring until all chitosan was dissolved.

(b) 3 grams of a reducing monosaccharide (e.g., glucose, galactose,ribose), or an equivalent amount of a reducing oligosaccharide, wasadded to the chitosan solution with gentle stirring. When the suspensionis homogeneous, 0.25 ml of toluene was added, and the flask was sealedwith aluminum foil. The magnetic stirring continued for 24 hours at roomtemperature.

(c) After stirring, the suspension was placed in a ventilated fume hoodwhere 1.327 grams of sodium borohydride in 5 ml of 0.1 M sodiumhydroxide was added in 0.2 ml aliquots (to avoid excessive foaming) toreduce Schiff bases and Amadori products. The solution was then coveredloosely with foil, stirred for 10 minutes at room temperature and then50 minutes in an ice bath.

(d) The flask was removed from the ice bath and the solution wasacidified to a pH of 5.5 by the dropwise addition of glacial acetic acid(approximately 1.9 ml) under further magnetic stirring to decomposeexcess borohydride.

(e) The solution was transferred to a dialysis bag (for example, aSPECTRA/POR #1 membrane obtained from Spectrum Medical Industries,Inc.). The solution was dialyzed against de-ionized water (4 liters×3)refrigerated (4° C.) over a period of 16 hours.

(f) The finished dialysate was transferred to a clean bottle and storein the refrigerator.

EXAMPLE 2

A solid weight determination may be made by transferring approximately5.0 ml of the glycated chitosan (GC) solution to a polystyrene weighingboat and placing it in a convection oven (temperature set at 60° C.) fordrying (which occurs in approximately 30 minutes). Record the following:Grams Weighing Boat (WB) WB + GC Solution, before drying WB + GCSolution, after drying

The percent solid weight of GC in the solution is calculated as follows:$\frac{\left( {{WB} + {{GC}\quad{solid}}} \right) - ({WB})}{\left( {{WB} + {{GC}\quad{Solution}}} \right) - ({WB})} \times 100\%$

EXAMPLE 3

For use, the GC solution may be sterilized. To achieve sterilization,quantities of the GC solution are placed in amber serum bottles whichare capped with butyl stoppers and sealed with an aluminum seal. Thebottles are then transferred to an autoclave, wherein the temperature isset to approximately 121° C. Sterilization is achieved in about 5minutes. Once the bottles have cooled to room temperature, the GCsolution is ready for use.

EXAMPLE 4

Particularly preferred for use in connection with the present inventionis galacto-chitosan. The structure of galacto-chitosan and thepreparation of galacto-chitosan from chitosan and D-galactose byreductive amination is shown diagrammatically below to furtherillustrate the best mode for preparing the most preferred form ofglycated chitosan:

EXAMPLE 5

Various solutions of galacto-chitosan were prepared in accordance withthe steps of Example 1 using as a source of chitosan PROTASAN UP CL110(chitosan chloride) obtained from Pronova Biopolymer which possessesapproximately 87% deacetylation and is of an average molecular weight ofabout 111,000 Daltons.

Example 5A

A solution was prepared wherein 30% of the free amino groups of thechitosan molecules (i.e., 30% of the 87%) were glycated with galactose.The resulting galacto-chitosan product possessed an average molecularweight of 128,130 Daltons.

Example 5B

A solution was prepared wherein 60% of the free amino groups of thechitosan molecules were glycated with galactose. The resultinggalacto-chitosan product possessed an average molecular weight of146,766 Daltons.

Example 5C

A solution was prepared wherein 90% of the free amino groups of thechitosan molecules were glycated with galactose. The resultinggalacto-chitosan product possessed an average molecular weight of165,181 Daltons.

EXAMPLE 6

Various solutions of galacto-chitosan were prepared in accordance withthe steps of Example 1 using as a source of chitosan a 21.4%deacetylated chitosan product (FLUKA Medium) obtained from Sigma-AldrichCo. which is of an average molecular weight of about 750,000 Daltons.

Example 6A

A solution was prepared wherein 30% of the free amino groups of thechitosan molecules (i.e., 30% of the 21.4%) were glycated withgalactose. The resulting galacto-chitosan product possessed an averagemolecular weight of 780,038 Daltons.

Example 6B

A solution was prepared wherein 60% of the free amino groups of thechitosan molecules were glycated with galactose. The resultinggalacto-chitosan product possessed an average molecular weight of809,660 Daltons.

Example 6C

A solution was prepared wherein 90% of the free amino groups of thechitosan molecules were glycated with galactose. The resultinggalacto-chitosan product possessed an average molecular weight of839,647 Daltons.

Soluble forms of glycated chitosan may be used individually or incombination, both as such and/or after additional chemical or enzymaticmodification. These modifications may include, but are not limited to,the generation of reactive groups such as carbonyls and carboxyl groupson the substituents introduced by glycation.

Aldehydes may be generated by oxidation of the carbohydrate side chain(e.g. treatment with periodate or lead tetraaceate) or, for example, theenzymatic oxidation of the primary alcohol group of galactosyl residueswith galactose oxidase.

Oxidation of the aldehyde groups (e.g. by treatment with hypohalites)may be utilized to obtain the carboxylic acid derivatives.Alternatively, bifunctional compounds containing both free carbonyl andcarboxylic groups (e.g. uronic acids) may be utilized during theglycation reaction.

Chitosan deamination with nitrous acid generates reducing aldoses andoligosaccharides suitable for the glycation of chitosan. Deamination ofthe deacetylated glucosaminyl residues by nitrous acid results in theselective cleavage of their glycosidic bonds with the formation of2,5-anhydro-D-mannose residues. Depending on the composition of specificareas of the chitosan chain, the anhydro hexose could be released as themonosaccharide, or occupy the reducing end of an oligosaccharide.Release of free N-acetylglucosamine could also occur from some regionsof the chitosan chain. Similar treatment of N-deacetylated glycoproteinsand glycolipids can be utilized to obtain oligosaccharides of definedchemical composition and biological activity for special preparations ofglycated chitosan. This includes normal as well as pathologicalglycoconjugates.

The various products obtained by chemical or enzymatic modification ofchitosan glycation may be reacted with other natural or syntheticmaterials, e.g., reaction of aldehyde-containing derivatives of glycatedchitosan with substances containing free amino groups, such as on theside chains of amino acids or proteins rich in lysine residues such asin collagen, on hexosamine residues as in chitosan and deacetylatedglycoconjugates, or on natural and synthetic amines and polyamines. Withdi- and poly amines this is expected to generate crosslinking throughSchiff base formation and subsequent rearrangements, condensation,dehydration, etc.

Stabilization of modified glycated chitosan materials can be made bychemical reduction or by curing involving rearrangements, condensationor dehydration, either spontaneous or by incubation under variousconditions of temperature, humidity and pressure.

The chemistry of Amadori reaarangements, Schiff bases and theLeukart-Wallach reaction is detailed in The Merck Index, Ninth Edition(1976) pp. ONR-3, ONR-55 and ONR-80, Library of Congress Card No.76-27231, the same being incorporated herein by reference. The chemistryof nucleophilic addition reactions as applicable to the presentinvention is detailed in Chapter 19 of Morrison and Boyd, OrganicChemistry, Second Edition (eighth printing 1970), Library of CongressCard No. 66-25695, the same being incorporated herein by reference.

The preferred viscoelastic solutions having greater than 1 wt. %glycated chitosan are especially useful as a tissue void replacementmaterial, such as for ophthalmic surgery, and as a lavage solution forpreventing abdominal adhesions following surgery. In a particularlypreferred aspect of the invention, a 9 wt. % aqueous solution ofglycated chitosan of a viscosity of about 77,000 centistokes is providedfor these purposes. In these applications, the inventive solutionssatisfy the need for a lightweight material for filling tissue voidswhile providing protection to tissues and contributing to hemostasis.

It will also be understood by those skilled in the art that theinventive viscosurgical preparations may be used in admixture with otherviscosurgical materials. For example, the invention glycated chitosansolutions are compatible with hyaluronic acid, chondroitin sulfate andCMC, whereby mixtures possessing desirable combinations of rheology,stability and biocompatibility may be achieved.

On the other hand, low viscosity solutions of glycated chitosan, i.e.,of a concentration less than 1.0 weight percent, and preferably between0.1 and 0.5 wt. %, and having a viscosity of between about 10 and 100centistokes, have application as an eye wetting agent/lubricant wherein.The product, being of a relatively high molecular weight, binds to thesurface of the eye, is non-toxic, and is absorbable.

In either high or low viscosity solutions, another aspect of theinvention is the ability of the inventive solutions to act as a drug ormedicament carrier. Drug molecules become entrapped in the glycatedchitosan solutions and thereby have the characteristic of sustainedrelease. The inventive solutions may be used effectively for both theprolongation and the control of the effective action of a drug ormedicament.

An important and surprising aspect of the invention is the discoverythat the inventive preparations of glycated chitosan unexpectedlypossess significant antimicrobial activity. The inventive preparationshave been tested and proved effective against common strains of bacteriaas indicated below:

EXAMPLE 7

For each indicated organism, media plates were inoculated such that auniform lawn of microorganisms would result. A sample of the inventivepreparation was placed on the surface of the media previouslyinoculated. The plates were incubated for 24-48 hours and examined forzones of inhibition extending beyond the edge of the sample. The sampleswere removed and the plates re-incubated to determine the viability ofthe microorganisms under the sample. Zone of Inhibition OrganismExtending Beyond Edge Beneath Sample Pseudomones aeruginosa not presentno growth Staph aureus not present no growth E. coli not present nogrowth Candida albicans not present no growth

While the benefit in connection with the foregoing preparations, theantimicrobial properties of the glycated chitosan opens the door to ahost of additional applications. For example, a low cost solution isprovided as an antimicrobial coating for adhesive strips and burn orwound dressings. In these applications, the inventive preparationsprovide the further benefits of hemostasis and an almost infinite shelflife. Inventive solutions may be used to coat or drench appliques or maybe applied directly to affected areas in liquid or powder form. In oneaspect of the invention, thin films of glycated chitosan are providedwhich may be incorporated into a wound dressing or which may otherwisebe used in medical applications, for example, as a natural polymericsubstrate to separate tissues for the prevention of tendon and ligamentadhesions after orthopedic surgery and for guided tissue regeneration indental surgery. Such films may be produced by drying a stock solution ofglycated chitosan.

EXAMPLE 8

Small squares of a dry glycated chitosan film were prepared bydepositing 10 ml of a 1 wt. % solution of glycated chitosan on a 5 cm×5cm grid, then drying the preparation in an oven at 57° C.

In other aspects of the invention, the antimicrobial glycated chitosanpreparations may be combined with typical ingredients to formcosmetic/hygenic products such as mouth rinses, facial lotions and handand body disinfectants.

While the invention has been described with a certain degree ofparticularity, it is understood that the invention is not limited to theembodiment(s) set for herein for purposes of exemplification, but is tobe limited only by the scope of the attached claim or claims, includingthe full range of equivalency to which each element thereof is entitled.

1-14. (canceled)
 15. An eye drop preparation comprising less than 1percent by weight of a glycated chitosan polymer dispersed in an aqueoussolution, said aqueous solution having a viscosity of between 10-100centistokes measured at 25° C.
 16. A dried film comprising glycatedchitosan.
 17. A medicament for providing the sustained release of atherapeutic substance, comprising glycated chitosan having entrappedtherein a desired amount of said therapeutic substance.
 18. A method ofpreparing ophthalmic wetting agents for topical application to an eyecomprising the steps of: preparing an aqueous solution having less than1.0 percent by weight of glycated chitosan and 30% to 90% glycation ofotherwise free amino groups of said glycated chitosan; wherein saidaqueous solution has a viscosity from about 10 centistokes toapproximately 100 centistokes.
 19. A method of preparing ophthalmicwetting agents according to claim 18 wherein said aqueous solutioncomprises 0.01% to 0.5% by weight of glycated chitosan.
 20. A method ofpreparing sustained release ophthalmic wetting agents according to claim18 wherein said aqueous solution comprises 60% glycation of otherwisefree amino groups of said glycated chitosan.
 21. A method of preparingsustained release ophthalmic wetting agents for topical application toan eye according to claim 18 further comprising the steps of: applyingsaid preparation to an eye; allowing enzymes in eye secretions to breakdown said aqueous solution for providing characteristics of sustainedrelease.